Characterization of phosphatidylinositol-specific phospholipase C defects associated with thrombin-induced mitogenesis.
In a previous paper (Rath, H. M., Doyle, G. A. R., and Silbert, D. F. (1989) J. Biol. Chem. 264, 13387-13390), we reported a selection for the isolation of Chinese hamster lung fibroblasts (CCL39) defective in thrombin-induced mitogenesis. One mutant, D1-6b, had decreased production of inositol phosphates when challenged with activators of phosphatidylinositol turnover and extracts of this mutant showed a marked decrease in phospholipase C (PLC) activity toward phosphatidylinositol. In the current studies, the PLC activities of wild type CCL39 and D1-6b cytosolic extracts are further characterized. Wild type cytosol had at least two phosphatidylinositol-specific PLC isoenzymes, which could be separated by anion exchange chromatography and behaved differently in thermal inactivation studies. Since gel filtration of PLC activity in wild type extracts gave Mr values similar to that of previously characterized PLCs (140,000-200,000), immunoblots with antibodies to bovine brain isoenzymes were used to show that the PLC activities obtained by anion exchange chromatography were PLC-delta and PLC-gamma. Immunoblots with mutant D1-6b cytosol confirmed the presence of the PLC-gamma but showed no detectable PLC-delta. This activity in the mutant extracts eluted at the same conductivity on anion exchange columns and had the same kinetics of thermal inactivation as the PLC-gamma found in the wild type extracts. PLC-gamma from mutant extracts was active in assays containing phospholipid detergent mixed micelles but not in assays utilizing phospholipid vesicles, in sharp contrast to PLC-gamma from CCL39 extracts, which was active under either condition. Thus, the phosphatidylinositol-specific phospholipase C activity of mutant D1-6b is diminished both by the loss of PLC-delta and by the compromised behavior of PLC-gamma.
Rath
H M
HM
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Fee
J A
JA
Rhee
S G
SG
Silbert
D F
DF
D000818:Animals; D002455:Cell Division / Q000187:drug effects*; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000201:enzymology; D015151:Immunoblotting; D007700:Kinetics; D008081:Liposomes; D008168:Lung; D008970:Molecular Weight; D009154:Mutation; D010716:Phosphatidylinositols / Q000378:metabolism*; D011489:Protein Denaturation; D013816:Thermodynamics; D013917:Thrombin / Q000494:pharmacology*; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*
D008081:Liposomes; D010716:Phosphatidylinositols; D010738:Type C Phospholipases; D013917:Thrombin